Journal Pre-proof

Basophil reactivity to BNT162b2 is mediated by PEGylated lipid nanoparticles in PEG

allergic patients

Dr Alexander Troelnikov, MBBS, BMedSci (Hons), Griffith Perkins, BSc (Adv), Dr

Chino Yuson, MD FRACP, Aida Ahamdie, RN BN GDip(AC) Mn ScNP, Dr Summaya
Balouch, MBBS, Dr Plinio R Hurtado, MD, FRACP(eq), PhD, Dr Pravin Hissaria, MD,
MBBS, FRACP, FRCPA

PII: S0091-6749(21)00731-4
DOI: https://doi.org/10.1016/j.jaci.2021.04.032
Reference: YMAI 15106

To appear in: Journal of Allergy and Clinical Immunology

Received Date: 13 April 2021

Revised Date: 22 April 2021
Accepted Date: 28 April 2021

Please cite this article as: Troelnikov A, Perkins G, Yuson C, Ahamdie A, Balouch S, Plinio R Hurtado
Hissaria P, Basophil reactivity to BNT162b2 is mediated by PEGylated lipid nanoparticles in PEG
allergic patients, Journal of Allergy and Clinical Immunology (2021), doi: https://doi.org/10.1016/
j.jaci.2021.04.032.

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© 2021 Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma &
Immunology.
 1 Basophil reactivity to BNT162b2 is mediated by PEGylated lipid nanoparticles in

2 PEG allergic patients

4 Dr Alexander Troelnikov†

5 MBBS, BMedSci (Hons)

6 Immunology Registrar

7 SA Pathology, Adelaide, South Australia 5000

8 E: alexander.troelnikov@sa.gov.au

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9 P: +61 8 82047201

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11 Griffith Perkins†
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12 BSc (Adv)
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13 SA Pathology, Adelaide, South Australia 5000

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14 School of Biological Sciences, University of Adelaide, Adelaide, South Australia

15 5000
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16 E: griffith.perkins@adelaide.edu.au
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17 P: +61 8 83130138

18

19 Dr Chino Yuson

20 MD FRACP

21 Consultant Immunologist

22 Immunology Department, Royal Adelaide Hospital, Adelaide, South Australia 5000

23 E: chino.yuson@sa.gov.au

24 P: +61 8 7074 2880

25

Troelnikov, Perkins, Yuson et al. 1 Revised – Submission 21 April 2021

26 Aida Ahamdie

27 RN BN GDip(AC) Mn ScNP

28 Nurse Consultant

29 Immunology Department, Royal Adelaide Hospital, Adelaide, South Australia 5000

30 E: Aida.Ahmadie@sa.gov.au

31 P: +61 8 7074 2880

32

33 Dr Summaya Balouch

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34 MBBS

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35 Medical Officer

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Immunology Department, Royal Adelaide Hospital, Adelaide, South Australia 5000
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37 E: summaya.balouch@sa.gov.au
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38 P: +61 8 7074 2880

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40 Dr Plinio R Hurtado
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41 MD, FRACP(eq), PhD

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42 Immunologist

43 Department of Renal Medicine, Royal Adelaide Hospital, Adelaide, South Australia

44 5000

45 School of Medicine, University of Adelaide, Adelaide, South Australia 5000

46 E: plinio.hurtado@sa.gov.au

47 P: +61 431697737

48

49 Dr Pravin Hissaria

50 MD, MBBS, FRACP, FRCPA

Troelnikov, Perkins, Yuson et al. 2 Revised – Submission 21 April 2021

51 Senior Staff Immunologist and Immunopathologist

52 Immunology Department, Royal Adelaide Hospital, Adelaide, South Australia 5000

53 SA Pathology, Adelaide, South Australia 5000

54 E: pravin.hissaria@sa.gov.au

55 P: +61 8 70740000

56
†
57 Co-first author

58

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59 Corresponding Author: Dr Pravin Hissaria

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Conflicts of interest: -p
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63 None of the authors declare any conflicts of interest.
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65 Authorship:
66 Alexander Troelnikov and Griffith Perkins contributed equally to the manuscript in
67 conception, design of the study, data analysis and writing of the manuscript and are co-
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68 first authors. Pravin Hissaria contributed to the conception, design and writing of the
69 manuscript. Chino Yuson, Summaya Balouch, Aida Ahamdie and Griffith Perkins
70 participated in acquisition of data. Plinio Hurtado contributed substantially to conception
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71 of the study and review of manuscript.

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Troelnikov, Perkins, Yuson et al. 3 Revised – Submission 21 April 2021

 72 Capsule Summary:
73 Polyethylene glycol (PEG) allergic patients demonstrate positive skin and basophil
74 activation tests to PEGylated liposomal drugs, BNT162b2 vaccine and doxorubicin, but
75 not to PEG alone, suggesting that presentation of PEG on PEGylated liposomes
76 facilitates hypersensitivity.
77
78 Words: 35
79
80 Clinical Implications:
81  Skin testing with BNT162b2 (Comirnaty) vaccine itself, and not PEG-containing

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82 surrogates, is required for diagnosis of BNT162b2 hypersensitivity
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83 Basophil activation tests to PEGylated liposomal drugs may be useful for the
84 assessment of BNT162b2-associated anaphylaxis
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89 Abstract
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Background:
92 The mechanisms underpinning allergic reactions to the BNT162b2 (Pfizer) COVID-19
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93 vaccine remain unknown, with polyethylene glycol (PEG) contained in the lipid
94 nanoparticle suspected as being the cause.
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95 Objective:
96 To evaluate the performance of skin testing and basophil activation testing to PEG,
97 polysorbate 80, BNT162b2 and AZD1222 (Astra-Zeneca) COVID-19 vaccines in patients
98 with a history of PEG allergy.
99 Methods:
100 Three known PEG allergic participants and three healthy controls were recruited, and
101 evaluated for hypersensitivity to the BNT162b2 and AZD1222 vaccines and related
102 compounds by skin testing and basophil activation measured by CD63 upregulation
103 using flow cytometry.
104 Results:
105 We found that the BNT162b2 vaccine induced positive skin tests in PEG allergic
106 patients, whereas traditional PEG skin testing was negative in two of three patients. One

Troelnikov, Perkins, Yuson et al. 4 Revised – Submission 21 April 2021

107 patient was found to be co-sensitized to both the BNT162b2 and AZD1222 vaccines due
108 to cross-reactive PEG and polysorbate allergy. The BNT162b2 vaccine, but not PEG
109 alone, induced dose-dependent activation of all patients’ basophils ex vivo. Similar
110 basophil activation could be induced by PEGylated liposomal doxorubicin, suggesting
111 that PEGlyated lipids within nanoparticles, and not PEG in its native state, are able to
112 efficiently induce degranulation.
113 Conclusions:
114 Our findings implicate PEG, as covalently modified within the vaccine lipid nanoparticle,
115 as a potential trigger of anaphylaxis to BNT162b2.
116

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117 Words: 210

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Troelnikov, Perkins, Yuson et al. 5 Revised – Submission 21 April 2021

120 Body Text

121 Introduction

122 Early safety monitoring during the mass vaccination campaigns internationally reported

123 an unexpectedly high rate of immediate hypersensitivity reactions to both the BNT162b2

124 (Comirnaty; Pfizer-BioNTech) and mRNA-1273 (Moderna) mRNA lipid nanoparticle

125 vaccines (1,2).

126

127 Although adverse event reporting more recently indicates a lower rate of anaphylaxis of

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128 4-5 per million for the BNT162b2 vaccine, in a well-characterised cohort of 64,900 health

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129 care workers, anaphylaxis was confirmed in 16 patients, or approximately 1 in every

130
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4000 individuals vaccinated (3), outnumbering the expected rate of 1 per million(4).
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131 Polyethylene glycol (PEG) conjugated to lipids, within the lipid nano-particle
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132 encapsulating the mRNA, is considered the likely culprit allergen (5). PEG driving the
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133 unanticipated frequency of anaphylaxis is surprising as this is otherwise an infrequent

134 cause of anaphylaxis despite its widespread presence in many other drugs, foods and
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135 cosmetics (6). To assess the risk for allergic reactions to BNT162b2, major authorities
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136 have suggested skin testing protocols for patients with a history of anaphylaxis to

137 vaccine components, in particular to PEG allergy and polysorbates (1,7,8). There have

138 been reports of cross-reactivity between PEG and polysorbates, and the AZD1222

139 vaccine, the only alternative COVID-19 vaccine currently available to our patients in

140 Australia, contains polysorbate 80 (1). This study aimed to evaluate a protocol for PEG,

141 polysorbate and COVID-19 vaccine skin and basophil activation testing in patients with

142 confirmed PEG hypersensitivity. The testing panel used is based on the presence of

143 PEG 2000 in the BNT162b2 and mRNA-1273 vaccines, and polysorbate 80 in AZD1222.

144

Troelnikov, Perkins, Yuson et al. 6 Revised – Submission 21 April 2021

145 Results and discussion

146 We recruited three adult patients known to our service with a history of PEG allergy

147 (demographics and index reaction details provided in Table 1). In addition to PEG,

148 Patient 1 had a history of allergy to polysorbates. Skin prick testing (SPT) and

149 intradermal testing (IDT) to PEG and polysorbates was performed using a panel adapted

150 from Banerji et al (1) (Table 1). The panel consisted of freshly thawed BNT162b2

151 (Comirnaty) and AZD1222 vaccines that were otherwise destined for discard, and readily

152 available and validated skin testing reagents that are related to components of the

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153 vaccines licensed for in vivo use (relevant component specified in Table 1). For the

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154 study, three healthy individuals used as controls were simultaneously tested with the skin

155
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testing panel. All patients developed a positive skin test to the BNT162b2 vaccine, whilst
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156 only Patient 1 tested positive for AZD1222, in agreement with the previous positivity skin
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157 test to polysorbate. Surprisingly, Patient 2 and Patient 3 had otherwise negative skin
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158 testing to the panel, including to PEG containing steroid methylprednisolone acetate at

159 1:10 dilution, the recommended, non-irritating concentration(9).

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160
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161 To further explore the results of the skin testing, we performed a basophil activation test

162 (BAT) with a range of PEG molecular weights (200 to 6000 g/mol) (Tokyo Chemical

163 Industry, Japan) and with both BNT162b2 vaccine and AZD1222. Prior to skin testing,

164 blood was drawn for basophil activation test (BAT) as previously described (10). In brief,

165 IL-3 treated whole blood was incubated with varying concentrations of allergen to induce

166 degranulation, and then stained with an antibody cocktail as described. Basophil

167 degranulation following allergen exposure was determined by surface expression of

168 CD63 and results compared to a positive (anti-IgE) and negative control (PBS). We

169 observed no increase in CD63 expression on basophils with PEGs of any molecular

Troelnikov, Perkins, Yuson et al. 7 Revised – Submission 21 April 2021

170 weight or with the AZD1222 vaccine (Table 2). However, we observed a dose-dependent

171 increase in CD63 expression in the presence of varying concentrations of BNT162b2 in

172 all three patients (figure 1), not observed in vaccinated nor unvaccinated, non-allergic

173 controls (n=3).

174

175 Given that the PEG-containing vaccine, but not PEG alone, was able to induce basophil

176 activation and skin test positivity, we repeated BAT with freshly prepared PEGylated

177 liposomal doxorubicin, which contains PEGylated liposomal nanoparticles similar to

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178 BNT162b2(11). We found similar dose dependent CD63 upregulation, even in the

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179 presence of very low concentrations of PEGylated liposomal doxorubicin (Figure 2; Table

180
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2) which was not observed in healthy controls, further supporting the contribution of the
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181 PEGylated liposomes to basophil activation.
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183 This is the first study to demonstrate positive COVID-19 vaccine allergy skin testing in a

184 well-characterized cohort of PEG allergic patients and validates specific vaccine skin
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185 testing for detection of PEG allergy. However, our study also demonstrates the
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186 limitations of current skin testing panels for macrogol and methylprednisolone acetate in

187 PEG diagnosis and indeed do not substitute for vaccine specific skin testing. Although all

188 of our patients had past positive PEG skin testing, current skin testing results suggested

189 that in those patients with historical reactions, intradermal testing with drugs containing

190 PEG excipients may yield false negatives. Intriguingly, BNT162b2 skin testing was able

191 to identify PEG allergy in all three PEG allergic patients despite two having otherwise

192 tested negative to PEG containing substances. A recent report also highlights this

193 limitation, with a single patient suffering vaccine allergy demonstrating skin test positivity

194 to only PEG 4000 but not to other molecular weight PEGs and PEG-containing

Troelnikov, Perkins, Yuson et al. 8 Revised – Submission 21 April 2021

195 substances (12). Our results indicate a need to re-evaluate skin testing protocols for

196 suspected PEG allergy and to include PEGylated liposomal compounds.

197

198 Current recommendations for PEG allergic patients suggest the use of an alternative

199 vaccine, however, in Patient 1, who was co-sensitized to polysorbate 80 and PEG, cross-

200 reactivity between BNT162b2 and AZD1222 vaccines was observed upon skin testing.

201 Co-sensitization with polysorbates may be seen in as many as 30% of PEG allergic

202 patients(13) and this would greatly limit COVID-19 vaccine options, given the presence

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203 of either excipient in all major vaccines in development(5). Given the urgent need for

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204 COVID-19 vaccination, it may be necessary to utilize a desensitization protocol for co-

205 sensitized patients.

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207 Ex vivo diagnosis of PEG allergy has historically been challenging and fraught with
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208 technical limitations (14,15) with no clear established protocols. Basophil activation

209 testing is a powerful diagnostic and prognostic allergy tool, providing complementary
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210 diagnostic information to specific IgE and skin testing, with superior diagnostic accuracy
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211 for certain drug and food allergies(16). In this study, basophil activation testing using

212 PEGylated liposomal nanoparticles was able to identify PEG allergic patients, even in the

213 absence of concurrent positive skin testing to PEG alone. We were able to show dose-

214 dependent basophil activation with BNT162b2 as well as PEGylated liposomal

215 doxorubicin, but not to unconjugated PEG. Allergic reactions to PEGylated

216 chemotherapeutics have long been known to cause hypersensitivity like reactions,

217 although they have been purported to be non-IgE-mediated, complement activation

218 related pseudo-allergy (CARPA) reactions (17). Some authors have suggested that

219 CARPA may be responsible for COVID-19 PEGylated lipid nanoparticle vaccine related

Troelnikov, Perkins, Yuson et al. 9 Revised – Submission 21 April 2021

220 allergy (1,18). In our study, the concurrent pattern of positive and negative skin and

221 basophil activation tests suggest that the PEG conformation or arrangement on the

222 surface of nanoparticles determines activation of basophils. This could be the result of

223 increased avidity augmenting IgE crosslinking on the surface of basophils, or by

224 facilitating oligomerization-dependent, IgG-mediated complement activation(19). Future

225 studies are required to determine the exact mechanism underlying hypersensitivity in

226 these patients. Our study also demonstrates that PEGylated doxorubicin can effectively

227 activate these patients’ basophils indicating that other PEGylated drugs could be

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228 surrogates to detect significant sensitization to PEGylated lipid nanoparticles. By

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229 contrast, AZD1222 did not induce ex vivo basophil activation in the patient with positive

230 skin testing.

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232 Small sample size is a limitation of our study, however we believe that our findings
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233 emphasize the importance of validating PEG allergy testing protocols in the assessment

234 of suspected allergic reactions to PEGylated drugs, including COVID-19 vaccines.

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235
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236 This is the first report implicating PEGylated lipids as mediators of allergy to BNT162b2

237 vaccine, as demonstrated by intradermal and basophil activation tests. Our study

238 indicates the indispensable requirement for specific vaccine skin testing in the evaluation

239 of patients following BNT162b2 allergic reactions. Further studies are required to clarify

240 whether these findings hold true in patients who experience allergic reactions following

241 administration of the COVID-19 vaccines.

242

243 Words: 1299

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Troelnikov, Perkins, Yuson et al. 10 Revised – Submission 21 April 2021

245 References

246 1. Banerji A, Wickner PG, Saff R, Stone CA, Robinson LB, Long AA, et al. mRNA

247 Vaccines to Prevent COVID-19 Disease and Reported Allergic Reactions: Current

248 Evidence and Suggested Approach. J Allergy Clin Immunol Pract. 2020 Dec;1–15.

249 2. Shimabukuro T, Nair N. Allergic Reactions Including Anaphylaxis After Receipt of

250 the First Dose of Pfizer-BioNTech COVID-19 Vaccine. JAMA. 2021 Jan

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252 3. Blumenthal KG, Robinson LB, Camargo CA, Shenoy ES, Banerji A, Landman AB,

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253 et al. Acute Allergic Reactions to mRNA COVID-19 Vaccines. JAMA. 2021 Mar 8;

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254 4. Su JR, Moro PL, Ng CS, Lewis PW, Said MA, Cano M V. Anaphylaxis after

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257 5. Castells MC, Phillips EJ. Maintaining Safety with SARS-CoV-2 Vaccines. Longo
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259 6. Stone CA, Liu Y, Relling M V., Krantz MS, Pratt AL, Abreo A, et al. Immediate
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261 We Have Recognized. J Allergy Clin Immunol Pract. 2019;7(5):1533-1540.e8.

262 7. Klimek L, Jutel M, Akdis CA, Bousquet J, Akdis M, Torres‐Jaen M, et al. ARIA‐

263 EAACI statement on severe allergic reactions to COVID‐19 vaccines – an EAACI‐

264 ARIA position paper. Allergy. 2020;0–3.

265 8. Turner PJ, Ansotegui IJ, Campbell DE, Cardona V, Ebisawa M, El-Gamal Y, et al.

266 Covid-19 Vaccine-Associated Anaphylaxis: a Statement of the World Allergy

267 Organization Anaphylaxis Committee. World Allergy Organ J. 2021;14(2):100517.

268 9. Li PH, Wagner A, Thomas I, Watts TJ, Rutkowski R, Rutkowski K. Steroid Allergy:

269 Clinical Features and the Importance of Excipient Testing in a Diagnostic

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270 Algorithm. J Allergy Clin Immunol Pract. 2018;6(5):1655–61.

271 10. Le TT (Adriana), Fok JS, Joseph SV, Eldi P, Chataway T, Smith W, et al.

272 Transplant induced food sensitization without allergy—mechanisms of tolerance. J

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274 11. Janssen. CAELYX ® Pegylated Liposomal Doxorubicin Hydrochloride for Injection

275 Sterile aqueous suspension for intravenous administration (2 mg/mL)

276 Antineoplastic Agent. 2018.

277 12. Sellaturay P, Nasser S, Islam S, Gurugama P, Ewan PW. Polyethylene glycol

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279 Clin Exp Allergy. 2021 Apr 6;(March):1–3.

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Bruusgaard-Mouritsen MA, Johansen JD, Garvey LH. Clinical manifestations and
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281 impact on daily life of allergy to polyethylene glycol (PEG) in ten patients. Clin Exp
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283 14. Schellekens H, Hennink WE, Brinks V. The immunogenicity of polyethylene glycol:

284 Facts and fiction. Pharm Res. 2013;30(7):1729–34.

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285 15. Zhou Z-H, Stone CA, Jakubovic B, Phillips EJ, Sussman G, Park J, et al. Anti-PEG
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286 IgE in anaphylaxis associated with polyethylene glycol. J Allergy Clin Immunol

287 Pract. 2021 Apr;9(4):1731-1733.e3.

288 16. Ebo DG, Bridts CH, Mertens CH, Sabato V. Principles, potential, and limitations of

289 ex vivo basophil activation by flow cytometry in allergology: A narrative review. J

290 Allergy Clin Immunol. 2021 Apr;147(4):1143–53.

291 17. Kozma GT, Mészáros T, Vashegyi I, Fülöp T, Örfi E, Dézsi L, et al. Pseudo-

292 anaphylaxis to Polyethylene Glycol (PEG)-Coated Liposomes: Roles of Anti-PEG

293 IgM and Complement Activation in a Porcine Model of Human Infusion Reactions.

294 ACS Nano. 2019;13(8):9315–24.

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295 18. Klimek L, Novak N, Cabanillas B, Jutel M, Bousquet J, Akdis CA. Allergenic

296 components of the mRNA-1273 vaccine for COVID-19: possible involvement of

297 polyethylene glycol and IgG-mediated complement activation. Allergy. 2021;0–2.

298 19. Strasser J, de Jong RN, Beurskens FJ, Wang G, Heck AJR, Schuurman J, et al.

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300 Activation on Antigenic Surfaces. Nano Lett. 2019 Jul 10;19(7):4787–96.

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302 Table 1: Patient characteristics and summarized skin test results. All skin tests were

303 carried out at as a series of three dilutions (1:1, 1:10 and 1:100) to a maximal, non-

304 irritating concentration as outlined in the table.

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306 Table 2: Summary of basophil activation test results. Results indicate the concentration

307 at which the percentage of CD63+ basophils is greater than 25 percent of the positive

308 control.

309

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310 Figure 1: Ex vivo basophil activation in response to the BNT162b2 (Comirnaty) vaccine.

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311 A: Gating strategy for basophils and basophil activation demonstrated for a positive

312
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response. CD63 positive gating set with fluorescence minus one (FMO).
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313 B: Activation of basophils from three patients and a representative healthy control in
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314 response to varied concentrations of BNT162b2 vaccine (or a positive or negative

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315 control) presented as histograms. BNT162b2 was diluted in phosphate buffered saline to

316 achieve testing concentrations.

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317 C: Grouped comparison of three PEG allergic patients and three healthy controls.
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318 Statistical significance identified by unpaired t-test for each concentration: *P < 0.05.

319

320 Figure 2: Activation of basophils from three patients and a representative healthy,

321 control in response to two dilutions of PEGylated liposomal doxorubicin (or a positive or

322 negative control) presented as histograms and bar charts.

Troelnikov, Perkins, Yuson et al. 14 Revised – Submission 21 April 2021

323 Table 1.
Patient characteristics Patient 1 Patient 2 Patient 3
Age 51 55 22
Gender Female Male Female
Macrogol containing
aperients, polysorbate Macrogol containing Parenteral steroid
Preceding allergen triggers
containing drugs, aperient preparation
cosmetics and foods
Time since last reaction 3 months 3 months 5 years
Skin prick testing

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Drug Component allergen
Movicol 100mg/ml PEG 3350 Positive 1 mg/mL Negative 100 mg/mL Negative 100 mg/mL

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Optive ® advanced Polysorbate 80 + Carmellose Negative neat Negative neat Negative neat

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Cellufresh ® Carmellose Negative neat Negative neat Negative neat
Methylprednisolone

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PEG 3350 Negative 40 mg/ml Negative 40 mg/mL Negative 40 mg/mL
acetate 40mg/mL

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Methylprednisolone
Excipient free Negative 40 mg/ml Negative 40 mg/mL Negative 40 mg/mL
succinate 40mg/mL

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Triamcinolone 10mg/mL Polysorbate 80 Negative 10 mg/mL Negative 10 mg/mL Negative 10 mg/mL
BNT162b2 100μg/mL PEGylated nanoparticle Negative 100 μg/mL Negative 100 μg/mL Negative 100 μg/mL
AZD1222 neat
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Polysorbate 80 Negative- neat Negative- neat Negative- neat
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Intradermal testing
Drug Component allergen
Methylprednisolone
PEG 3350 Positive 0.4 mg/mL Negative 4 mg/mL Negative 4 mg/mL
acetate 4 mg/mL
Methylprednisolone
Excipient free Positive 0.04 mg/mL Negative 4 mg/mL Negative 4 mg/mL
succinate 4 mg/mL
Triamcinolone 1mg/mL Polysorbate 80 Positive 0.1 mg/mL - -
Optive ® Advanced 1;10
Polysorbate 80 - Negative 1:10 dilution Negative 1:10 dilution
dilution
BNT162b PEGylated nanoparticle Positive- 1 μg/mL Positive- 1 μg/mL Positive- 1 μg/mL
AZD1222 Polysorbate 80 Positive 1:10 Negative 1:10 Negative 1:10
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Troelnikov, Perkins, Yuson et al. 15 Revised – Submission 21 April 2021

325 Table 2.

Range of allergen Basophil Activation Test Results

Compound
concentrations tested Patient 1 Patient 2 Patient 3
BNT162b2 0.05 μg/ml to 10 μg/mL 5 μg/mL 0.05 μg/mL 0.05 μg/mL
PEGylated Liposomal
1μg/ml to 10 μg/mL 10 μg/mL 1 μg/mL 10 μg/mL
Doxorubicin

AZD1222 1:2000 to 1:10 dilution Negative Negative Negative

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PEG2000 0.05 μg/mL to 5 mg/mL Negative Negative Negative

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PEG 200 5 mg/mL Negative Negative Negative

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PEG 400 5 mg/mL Negative Negative Negative

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PEG 600 5 mg/mL Negative Negative Negative

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PEG 6000 5 mg/mL Negative Negative Negative
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